Atherosclerosis (AS) is the principal cause of cardiovascular disease. AS is a disease of the intima of the arteries, especially of the large arteries, that leads to fatty lesions called atheromatous plaques on the inner surfaces of the arteries. The earliest stage in the development of these lesions is believed to be damage to the endothelial cells and sublying intima. The damage can be caused by physical abrasion of the endothelium, by abnormal substances in the blood, or even by the effect of the pulsating arterial pressure on the vessel wall. Once the damage has occurred, the endothelial and smooth muscle cells swell and proliferate and migrate from the media of the arteries into the lesion. Soon thereafter, lipid substances, especially cholesterol, begin to deposit from the blood in the proliferating cells, forming the atheromatous plaques.
In the later stages of the lesions, fibroblasts infiltrate the degenerative areas and cause progressive sclerosis (fibrosis) of the arteries. Still later, calcium often precipitates with the lipids to develop calcified plaques. When these two processes have occurred, the arteries are then extremely hard, and the disease is called arteriosclerosis, or simply "hardening of the arteries."
Arteriosclerotic arteries lose most of their distensibility, and because of the degenerative areas they are easily ruptured. Also, the atheromatous plaques of their surfaces causes blood clots to develop, with resultant thrombus or embolus formation. Almost half of all human beings in the United States and Europe die of arteriosclerosis. Approximately two thirds of these deaths are caused by thrombosis of one or more coronary arteries, and the remaining one third by thrombosis or hemorrhage of vessels in other organs of the body, such as in the brain which causes strokes, as well as in the kidneys, liver, gastrointestinal tract, and limbs.
An important part of this response to injury model in AS is the action of smooth muscle cells. Once the lumen of the vessel has been damaged by hypercholesterolemia, hypertension or some other pathological process, proliferation of smooth muscle cells occurs. This is followed by the formation of a connected tissue matrix, which comprises the groundwork for the atherosclerotic plaque. Smooth muscle cells are not only responsible for the formation of this matrix, but also contain the ability to express genes for a number of growth regulatory molecules, as well as receptors to growth factors. Thus, smooth muscle cells play a pivotal role in the pathogenesis of AS.
Homocysteine, a sulfur-containing amino acid, is an intermediate metabolite of methionine. Elevated plasma homocysteine, which results from an inherited disorder of methionine metabolism, is linked to an increased risk of cardiovascular disease. Homocystinuria is associated with the early onset of arteriosclerosis and frequent life-threatening thrombotic episodes, in which cerebral infarction is more common than myocardial infarction. Mudd, S. H., et al., (1985). "The natural history of homocystinuria due to cystathionine .beta.-synthase deficiency" Am. J. Hum. Genet. 37:1-31.
Animal studies have shown that homocysteine is a potent inducer of atherosclerosis, causing visible vascular damage within 1 week of continuous administration. Individuals with an inborn error of homocysteine metabolism have marked hyperhomocystinemia, mental retardation, and severe atherosclerosis that usually results in death by age 15. Numerous studies have documented the association between occlusive vascular disease and elevated blood levels of homocysteine.
While it is now generally agreed that homocysteine (Hcy) is an independent risk factor for atherosclerosis, there has been less agreement on the biological basis or mechanism of action of the atherogenic effect of Hcy. A growth effect of Hcy upon vascular smooth muscle has been shown with several different approaches. For example, hypertrophy, hyperplasia and migration of smooth muscle cells are the most prominent features of atherosclerotic arteries in miniature swine with experimental hyperhomocystinemia. This is consistent with the finding of a strong positive correlation between serum Hcy concentration and carotid artery wall thickness in man, with a major increase in medial thickness. With extreme elevation of serum Hcy (e.g., in homocystinuric patients who are homozygous for a mutant non-functional cystathione-.beta.-synthase gene), the resulting atheromata are primarily composed of vascular smooth muscle cells that produce a fibrotic lesion. These may progress to fibrolipid lesions when lipoproteins bind to the vessel matrix that is induced by Hcy. While the mechanism of this growth effect of Hcy is not well understood, there is evidence that Hcy is directly mitotic for aortic smooth muscle cells.
Tsai et al. recently have shown that Hcy is mitogenic for rat aortic smooth muscle cells in vitro, and referred to a "growth factor-like" activity of Hcy. Tsai, J. et al., (1994). "Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis," Proc. Natl. Acad. Sci., 91:6369-6373. They found that Hcy induces upregulation of cyclins. The inventors have now found that Hcy also has a mitogenic effect upon avian and mammalian aortic smooth muscle cells in vitro. The inventor's data showed a growth factor effect of Hcy that appears to be receptor-mediated. This finding has led to the discovery of a novel Hcy receptor that has anatomic and physiological properties that may account for a major effect of Hcy in atherogenesis. This receptor can be blocked by antagonists in the NMDA receptor antagonist family as a new and effective way of preventing and treating atherosclerosis. Further, cloning of this receptor will allow for the development of antagonists which are even more specific for the Hcy receptor.
It is therefore a primary objective of the present invention to provide a new means of preventing and treating atherosclerosis without the side effects associated with alcohol treatment.
It is a further objective of the present invention to provide a method of treating atherosclerosis using NMDA antagonists to block the growth factor effect of homocysteine.
It is yet a further objective of the present invention to provide a means of isolating and characterizing the Hcy NMDA-like receptor.
It is still a further objective of the present invention to provide a means of cloning the homocysteine NMDA-like receptor.
It is a further objective of the present invention to identify antagonists which are specific to the Hcy receptor.
These and other objectives will become clear from the foregoing detailed description.